Sweet-talk: role of host glycosylation in bacterial pathogenesis of the gastrointestinal tract.

Glycosylation is a key modification of proteins and lipids and is involved in most intermolecular and intercellular interactions. The gastrointestinal mucus gel is continuous and can be divided into two layers: a secreted loosely associated layer and a layer firmly attached to the mucosa. In addition, the membrane-bound glycosylated proteins and lipids create a glycocalyx, which remains adherent on each cell and is dynamic and responsive to the physiological state and environment of the cell. The secreted glycans form a mucus gel layer that serves as a physicochemical sensor and barrier network and is primarily composed of mucins and associated peptides. These glycans protect gut epithelial cells from chemical, biological and physical insults and are continuously renewed. Pathogens colonise and invade the host epithelial cells using protein-protein and glycan-lectin interactions. During the process of colonisation and infection, the glycosylation state of both host and pathogen change in response to the presence of the other. This complex modulation of glycan expression critically determines pathogenesis and the host response in terms of structural changes and immune response. In addition, by influencing host immunity and gut glycosylation, the microbiota can further effect protection against pathogens. In this review, the roles of host glycosylation in interactions with two prevalent bacterial pathogens, Campylobater jejuni and Helicobacter pylori, are discussed to illustrate important concepts in pathogenesis.

Triggering receptor expressed on myeloid cells-1 (TREM-1) expression on gastric epithelium: implication for a role of TREM-1 in Helicobacter pylori infection.

In Helicobacter pylori gastritis gastric epithelium plays a central role in the innate immunity to H. pylori. However, epithelial receptors interacting with H. pylori have been poorly characterized so far. Recently a new triggering receptor expressed on myeloid cells-1 (TREM-1) has been identified on human neutrophils and monocytes. On these cells TREM-1 triggers innate immunity by stimulating the secretion of interleukin (IL)-8 and tumour necrosis factor (TNF)-alpha and thus amplifies bacterial-induced inflammation. In this study expression and function of TREM-1 in gastric epithelium exposed to H. pylori has been investigated. TREM-1 mRNA and protein were expressed on gastric epithelial cell lines as demonstrated by reverse transcription-polymerase chain reaction (RT-PCR) and fluorescence activated cell sorter analysis. Gastric epithelial TREM-1 expression was up-regulated directly by H. pylori and was independent of epithelial IL-8 induced by H. pylori. Immunohistochemistry and tissue RT-PCR demonstrated significantly stronger TREM-1 expression in H. pylori gastritis compared with the non-inflamed gastric mucosa supporting in vivo that epithelial TREM-1 is up-regulated during H. pylori infection. Stimulation of gastric epithelial TREM-1 receptor resulted in IL-8 up-regulation on mRNA and protein level, as shown by real-time PCR and immunoassay. This is the first study localizing TREM-1 on gastric epithelium. Functional data suggest that TREM-1 expressed on gastric epithelium amplifies inflammation of the underlying gastric mucosa by up-regulation of IL-8.

Co-induction of anaesthesia with 0.75 mg kg propofol followed by sevoflurane: a randomized trial in the elderly with cardiovascular risk factors.

BACKGROUND: The induction of general anaesthesia is associated with the greatest cardiovascular changes in elderly patients. Induction can be performed either intravenously or with gaseous induction. Sevoflurane has advantages over propofol for induction of anaesthesia in the elderly, since the lower reduction in mean arterial pressure with sevoflurane is both statistically and clinically significant. This prospective randomized controlled trial investigated the cardiovascular benefits of co-induction of anaesthesia with 0.75 mg kg(-1) propofol and 8% sevoflurane, when compared with 8% sevoflurane alone in patients requiring surgery for fractured neck of femur. METHOD: In total, 38 patients aged 75 or over were allocated into the two groups, receiving either 0.75 mg kg(-1) of propofol followed by 8% sevoflurane or 8% sevoflurane alone. Vital signs were recorded until successful insertion of a laryngeal mask. Induction times, induction events and patient satisfaction scores were also recorded. RESULTS: Results showed that there were no differences in the cardiovascular parameters between the two groups. Induction times were faster in the propofol and sevoflurane group (62 vs. 81 s; P = 0.028). The postoperative questionnaire showed that the majority of patients in both groups were satisfied with the induction process. CONCLUSIONS: We concluded that 0.75 mg kg(-1) of propofol followed by sevoflurane induction is an acceptable alternative to sevoflurane induction. It is associated with similar haemodynamic variables, faster induction times and is very well tolerated.

Molecular mimicry between Helicobacter pylori and the host.

Helicobacter pylori lipopolysaccharide (LPS) expresses Lewis x and Lewis y blood group antigens that are identical to those occurring in the human gastric mucosa. During infection, antibodies against LPS, which bind to host Lewis antigens, may be induced. These consequently recognize gastric glycoprotein targets and cause autoimmune inflammation.

Why Helicobacter pylori has Lewis antigens.

In mimicry with human gastric epithelial cells, the lipopolysaccharide of Helicobacter pylori expresses Lewis blood group antigens. Recent data suggest that molecular mimicry does not promote immune evasion, nor does it lead to induction of autoantibodies, but that H. pylori Lewis X mediates adhesion to gastric epithelial cells and is essential for colonization.

Interaction of Campylobacter jejuni with extracellular matrix components.

The adhesion of three strains of Campylobacter jejuni to coverslips and microwells coated with isolated extracellular matrix components, fibronectin, laminin and types I, III, IV and V collagens was studied. Fibronectin mediated the adherence of C. jejuni, but there were differences in the binding capacities of the strains. Type I, III and V collagens mediated very strongly the attachment of two strains of C. jejuni. All three strains attached weakly to basement membrane-specific type IV collagen. Laminin was capable of mediating the adhesion only when present at a higher concentration. The observations indicate that extracellular matrix components may serve as anchor molecules for C. jejuni adhesion and that several attachment mechanisms occur simultaneously.

Lipopolysaccharides in the development of the Guillain-Barré syndrome and Miller Fisher syndrome forms of acute inflammatory peripheral neuropathies.

Guillain-Barré syndrome (GBS), an acute inflammatory polyneuropathy, is preceded in most cases by an infectious illness, and Campylobacter jejuni, a leading cause of acute gastroenteritis, is the most common antecedent to GBS and its ocular variant, Miller Fisher syndrome (MFS). O (Penner) serotyping is considered to distinguish between C. jejuni strains based on differences in lipopolysaccharide (LPS) structure. Serotypes of C. jejuni uncommon in enteritis, such as serotype O:19 and O:41, have been associated with GBS. Chemical studies on the core oligosaccharide (OS) of C. jejuni LPSs from serotypes including O:1, O:2, O:4, O:10, O:19, O:23, O:36 and O:41 have revealed structures that mimic human gangliosides including GM1, GD1a, GD2, GD3, and GM2. Research has focused on the view that molecular mimicry may be a factor in the pathogenesis of GBS. Serum antibodies against gangliosides, particularly GM1 ganglioside, are present in 30% of GBS patients, and are highly associated with MFS, but are generally absent in enteritis cases uncomplicated by neuropathy. Collective data from human and animal studies with anti-ganglioside antibodies suggest a pathogenic role for the antibodies. Many aspects of the pathogenesis of GBS are unclear, in particular how LPS is presented to T cells or the role of host factors in disease development.

Ganglioside-induced antiganglioside antibodies from a neuropathy patient cross-react with lipopolysaccharides of Campylobacter jejuni associated with Guillain-Barré syndrome.

Antiganglioside serum antibodies from a patient treated with gangliosides were examined for cross-reactivity with lipopolysaccharides (LPSs) of Campylobacter jejuni strains associated with Guillain-Barré syndrome (GBS). The patient had no preceding infection with C. jejuni and developed chronic progressive motor polyneuropathy following parenteral ganglioside treatment. Serum IgG antibodies recognised GM1 and GD1b gangliosides as well as asialo-GM1, and cross-reactivity was observed with LPSs from C. jejuni O:2, O:4, O:19 and O:41. The results give a clear indication that gangliosides and LPSs from C. jejuni serotypes associated with GBS share common epitopes.

Cytotoxicity associated with induction of nitric oxide synthase in rat duodenal epithelial cells in vivo by lipopolysaccharide of Helicobacter pylori: inhibition by superoxide dismutase.

The products released by Helicobacter pylori (H. pylori) in the gastric antral and duodenal mucosa may be involved in mucosal ulceration by stimulating the local formation of cytotoxic factors such as nitric oxide (NO), superoxide or peroxynitrite. The present study investigates the ability of purified H. pylori lipopolysaccharide (LPS) to induce nitric oxide synthase (iNOS) in rat duodenal epithelial cells following in vivo challenge and its interaction with superoxide in promoting cellular damage and apoptosis. H. pylori LPS (0.75-3 mg kg(-1) i.v. or 3-12 mg kg(-1) p.o.) induced a dose - dependent expression of iNOS activity after 5 h in the duodenal epithelial cells, determined by [(14)C] arginine conversion to citrulline. The epithelial cell viability, as assessed by Trypan Blue exclusion and MTT conversion, was reduced 5 h after challenge with H. pylori LPS, while the incidence of apoptosis was increased. The iNOS activity and reduction in cell viability following H. pylori LPS challenge i.v. was inhibited by the selective iNOS inhibitor, 1400 W (0.2-5 mg kg(-1) i.v.). Concurrent administration of superoxide dismutase conjugated with polyethylene glycol (250 - 500 i.u. kg(-1), i.v.), which did not modify the cellular iNOS activity, reduced the epithelial cell damage provoked by i.v. H. pylori LPS, and abolished the increased incidence of apoptosis. These results suggest that expression of iNOS following challenge with H. pylori LPS provokes duodenal epithelial cell injury and apoptosis by a process involving superoxide, implicating peroxynitrite involvement. These events may contribute to the pathogenic mechanisms of H. pylori in promoting peptic ulcer disease.

The role of lipopolysaccharide in Helicobacter pylori pathogenesis.

The present review describes the structure, attributes and properties of Helicobacter pylori lipopolysaccharides (LPS), and their potential role in pathogenesis. Although possessing certain attributes similar to those of LPS of other Gram-negative bacteria, H. pylori LPS possess unique biological properties. H. pylori LPS has, in general, low immunological activity and this property may aid the persistence of infection. The O-specific chain of the LPS mimics Lewis blood group antigens in structure. As these antigens are present in the gastric mucosa, the expression of Lewis antigens on the bacterial surface may camouflage the bacterium and aid survival of H. pylori. Alternatively, since autoantibodies against human antral gastric mucosa have been observed in H. pylori-positive patients, the relevance of LPS in the development of autoimmunity in H. pylori-associated disease requires further investigation. H. pylori LPS in part mediates the binding of the bacterium to laminin, and interferes with gastric cell receptor-laminin interaction, thereby potentially contributing to the loss of mucosal integrity. In vitro observations of inhibition of sulphated mucin synthesis and stimulation of pepsinogen secretion by LPS suggest new mechanisms for H. pylori-induced mucosal damage. Nevertheless, further in vivo studies are required to support their pathogenic role.

Serological analysis of the heat-stable antigens involved in serotyping Campylobacter jejuni and Campylobacter coli.

Analysis with serotyping antisera showed that carbohydrate determinants were the dominant heat-stable antigens of Campylobacter jenuni/coli involved, whereas proteins did not contribute to the serological reactions. Lipopolysaccharide (LPS) along with a polysaccharide extract from whole bacteria (PS(WB] conferred strain serospecificity. In general, analysis with monoclonal antibodies in passive haemagglutination and co-agglutination tests showed the existence of similar antigenic determinants in LPS and PS(WB) of the same strain. However, in some strains determinants were detectable in LPS but not in PS(WB) using monoclonal antibodies, in other strains the situation was reversed. All of these monoclonal antibodies reacted with LPS in the more sensitive immunoblotting technique. The presence of 2-keto-3-deoxyoctonic acid in PS(WB) preparations, in the absence of endotoxin, supported the conclusion that PS(WB) was derived from LPS during extraction. The lack of detection of a reaction by monoclonal antibodies with LPS in passive haemagglutination, in contrast to immunoblotting, was suggested due to the presence of low concentrations of the relevant epitopes because of the procedure used to prepare the LPS tested.

Comparison of three serological methods for detection of Lewis antigens on the surface of Helicobacter pylori.

Chemical studies have shown that lipopolysaccharide (LPS) from certain strains of Helicobacter pylori expresses the Lewis (Le) blood group antigens, predominantly Le(x) and/or Le(y). Serological methods have been used in a number of studies to assess the level of Le expression by H. pylori. However, serological and chemical analyses of a strain's LPS do not always agree. The purpose of this study was to evaluate and compare serological methods used to detect the expression of these antigens by H. pylori. A set of 34 Irish clinical isolates and three culture collection strains had their Le antigen expression assessed using three serological methods, serodot, Western blotting and enzyme-linked immunosorbent assay. A combination of all three methods was used to estimate Le(x) (59%) and Le(y) (94%) expression and this may allow a more accurate assessment of Le expression by isolates for pathogenesis studies.

Helicobacter pylori lipopolysaccharide provokes iNOS-mediated acute systemic microvascular inflammatory responses in rat cardiac, hepatic, renal and pulmonary tissues.

We have examined the effects of intravenous administration of a purified lipopolysaccharide (LPS) from Helicobacter pylori (3 mg kg(-1), i.v.) on rat vascular permeability, assessed by the radiolabelled human serum albumin leakage technique in the heart, kidney, liver and lung 4 h after challenge. An increased vascular permeability in cardiac, renal, hepatic and pulmonary tissues after challenge was determined. The albumin leakage observed in all these organs could be prevented by the selective inducible nitric oxide synthase inhibitor, N-(8-(aminomethyl)benzyl)-acetamidine (1400W; 0.2-1 mg kg(-1), s.c.) administered concurrently with LPS. Thus, H. pylori LPS can provoke a microvascular inflammatory response in the rat cardiac, renal, hepatic and pulmonary tissues, actions mediated through the activation of the inducible nitric oxide synthase isoenzyme.

Attenuation of Helicobacter pylori endotoxin-provoked rat intestinal inflammation by selective inhibition of the inducible nitric oxide synthase.

We studied the actions of purified Helicobacter pylori endotoxin (3 mg kg(-1), i.v.) on rat intestinal vascular permeability (assessed by the radiolabelled human serum albumin leakage technique) and on nitric oxide synthase induction (assessed by the citrulline assay) 4 h later. We found increased albumin leakage and expression of the inducible nitric oxide synthase in jejunum and colon, effects reversed by a selective inducible nitric oxide synthase inhibitor N-(8-(aminomethyl)benzyl)-acetamidine (1400W; 0.2-1 mg kg(-1), s.c., concurrently with endotoxin). Thus, H. pylori endotoxin seems to be capable of provoking an inflammatory response in the rat intestinal tissue. Systemic liberation of H. pylori endotoxin might possibly attenuate jejunal and colonic mucosal barrier function, a process mediated by the expression of the inducible nitric oxide synthase.

Helicobacter pylori lipopolysaccharide-provoked injury to rat gastroduodenal microvasculature involves inducible nitric oxide synthase.

The actions of a purified Helicobacter pylori lipopolysaccharide (3 mg x kg(-1), i.v.) on rat gastric antral and duodenal microvascular integrity (determined as radiolabelled albumin leakage) and the expression of the inducible nitric oxide (NO) synthase (iNOS; assessed by the citrulline assay) were investigated 4 h after challenge. Significant increases of albumin leakage and expression of iNOS in both antral and duodenal tissues were observed following challenge. Concurrent administration of the selective iNOS inhibitor, 1400W (N-(8-(aminomethyl)benzyl)-acetamidine; 0.2-1 mg x kg(-1), s.c.), with lipopolysaccharide, caused a dose-dependent attenuation of the gastric and duodenal albumin leakage. Thus, H. pylori lipopolysaccharide can initiate the expression of iNOS in the stomach and duodenum following systemic challenge, which can provoke gastroduodenal microvascular dysfunction.

Biological and serological characterization of Campylobacter jejuni lipopolysaccharides with deviating core and lipid A structures.

Lipopolysaccharides from Campylobacter jejuni were tested for their ability to induce toxic lethality in galactosamine-sensitized mice, pyrogenicity in rabbits and tumour necrosis factor (TNF) secretion from mouse peritoneal macrophages. Compared with those of Salmonella LPS, lethal toxicity was 50% lower, pyrogenicity was 30- to 50-fold lower, and ability to induce TNF was 100-fold lower. C. jejuni LPS and lipid A exhibited higher phase-transition temperatures than those of Salmonella preparations, and thus the former have lower fluidity at 37 degrees C. This lower fluidity of acyl chains may influence the biological activities of C. jejuni LPS, but acyl chain characteristics and diaminoglucose replacing glucosamine in the hydrophilic lipid A backbone may also influence the supramolecular structure of lipid A, thereby affecting biological activities. Although diaminoglucose is present in the backbone of C. jejuni lipid A, antigenically the latter resembled classical lipid A of the Enterobacteriaceae when tested with anti-lipid A antibodies. Chemical investigations suggested the presence of glucuronic acid in an acid labile linkage in the inner core region, thus producing a structurally unusual region in C. jejuni LPS.

Cell surface characteristics of Helicobacter pylori.

Helicobacter pylori is an important gastroduodenal pathogen of humans. Immunological and structural studies have been performed on the phospholipids, lipopolysaccharides (LPS) and some surface proteins of H. pylori strains. H. pylori LPS has, in general, low immunological activity and this property may aid the survival of this chronic infection. Nevertheless, H. pylori LPS has been found to influence the quality of gastric mucin and to stimulate pepsinogen secretion, thereby contributing to gastric disease. A number of putative adhesins of the bacterium have been described. This multiplicity of adhesins may reflect that H. pylori adherence is a multi-step process involving different interactions, and that different adhesins may mediate adherence to various sites in gastric tissue.

Molecular mimicry of host structures by bacterial lipopolysaccharides and its contribution to disease.

The core oligosaccharides of low-molecular-weight lipopolysaccharide (LPS), also termed lipooligosaccharide (LOS), of pathogenic Neisseria spp. mimic the carbohydrate moieties of glycosphingolipids present on human cells. Such mimicry may serve to camouflage the bacterial surface from the host. The LOS component is antigenically and/or chemically identical to lactoneoseries glycosphingolipids and can become sialylated in Neisseria gonorrhoeae when the bacterium is grown in the presence of cytidine 5'-monophospho-N-acetylneuraminic acid, the nucleotide sugar of sialic acid. Strains of Neisseria meningitidis and Haemophilus influenzae also express similarly sialylated LPS. Sialylation of the LOS influences susceptibility to bactericidal antibody, may decrease or prevent phagocytosis, cause down-regulation of complement activation, and decrease adherence to neutrophils and the subsequent oxidative burst response. The core oligosaccharides of LPS of Campylobacter jejuni serotypes which are associated with the development of the neurological disorder, Guillain-Barré syndrome (GBS), exhibit mimicry of gangliosides. Cross-reactive antibodies between C. jejuni LPS and gangliosides are considered to play an important role in GBS pathogenesis. In contrast, the O-chain of a number of Helicobacter pylori strains exhibit mimicry of Lewis(x) and Lewis(y) blood group antigens. The role of this mimicry remains to be investigated, but may play a role in bacterial camouflage, the induction of autoimmunity and immune suppression in H. pylori-associated disease.

The relationship between O-chain expression and colonisation ability of Helicobacter pylori in a mouse model.

The influence of lipopolysaccharide (LPS) O-polysaccharide chain production on the colonisation ability of Helicobacter pylori in four mouse models (NMRI, C57BL/6, CBA/Ca, and BALB/cA mice) was studied. H. pylori strains that produced smooth-form LPS (S-LPS) detectable in silver-stained electrophoretic gels colonised mice. In contrast, a laboratory-passaged strain G50 and the culture collection strain CCUG 17874 did not colonise mice; the former strain produced low amounts of O-chains only detectable in immunoblotting but not in silver-stained gels, whereas the latter produced rough-form LPS (R-LPS) without O-chains. Furthermore, a galE isogenic mutant, which produced R-LPS, did not colonise mice. However, after repeated broth culture, strains G50 and CCUG 17874 produced S-LPS detectable in silver-stained gels and were capable of colonising mice. Consistent with the production of O-chains, all colonising strains produced Lewis (Le) antigens, Le(x) and/or Le(y). Except for low expression of Le(y) by non-colonising G50, reflecting low production of O-chains, all other non-colonising strains and the galE mutant lacked expression of Le antigens consistent with their production of R-LPS. Lectin typing of strains supported these findings, and also showed that lectin types did not differ before and after colonisation. The low level of O-chain production and Le antigen expression by the non-colonising G50 may not be sufficient to aid colonisation. Examination of protein profiles of H. pylori strains before inoculation showed that protein expression was not significantly different between colonising and non-colonising strains. These results show that S-LPS production with O-chain expression is required by H. pylori for colonisation in a number of mouse models and that care should be taken with inoculating H. pylori strains that loss of O-chains does not occur during subculturing.

Effect of host Lewis and ABO blood group antigen expression on Helicobacter pylori colonisation density and the consequent inflammatory response.

The Lewis(b) blood group antigen has been implicated as a putative receptor for Helicobacter pylori in the gastric mucosa. Furthermore, an increased prevalence of duodenal ulcer was found in non-secretors and it has been suggested that secretor status may influence bacterial colonisation density. Other investigators have hypothesised that severity of antral gastritis may be related to colonisation density of the bacterium alone, and that a critical bacterial load is necessary for the development of duodenal ulcer. Our objectives were to investigate whether a relationship existed between host Lewis and ABO blood group phenotype and prevalence of H. pylori infection. In addition we investigated whether bacterial colonisation density and the ensuing inflammatory response was influenced by secretor status and ABO blood group phenotype. The Lewis and ABO blood group phenotype of 207 patients undergoing upper endoscopy was determined. Of these, 136 were secretors and 62 were nonsecretors. Forty-five percent of patients were infected with H. pylori. No significant association was found between H. pylori infection and expression of Lewis(a) or Lewis(b) blood group antigen. The mean histological density of H. pylori was 1.8 +/- 0.2 among non-secretors and 1.51 +/- 0.13 among secretors (P = 0.209), with a mean grade of lymphocytic infiltration significantly greater in H. pylori-infected non-secretors (2.23 +/- 0.123 vs 1.8 +/- 0.074; P = 0.003). In addition, blood group O non-secretors had a significantly higher grade of lymphocyte infiltration of their gastric mucosa compared to non-O non-secretors (2.53 +/- 0.133 vs 1.93 +/- 0.181, P = 0.027). These results suggest that although no in vivo relationship exists between H. pylori and preferential adhesion to the putative Lewis(b) receptor, bacterial colonisation and the ensuing inflammatory response may be influenced at least in part by host expression of ABO and Lewisa blood group antigens.